Method for screening and remediation of petroleum-contaminated soil bioremediation agent

Method for screening and remediation of petroleum-contaminated soil bioremediation agent

  • CN 102,021,132 A
  • Filed: 11/19/2010
  • Published: 04/20/2011
  • Est. Priority Date: 12/03/2009
  • Status: Active Grant
First Claim
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1. the screening and the restorative procedure of an oil-polluted soils biological restoration microbial inoculum is characterized in that comprising following step:

  • Step 1;

    the collection in oil degradation original inhabitants bacterium and halophilic bacterium bacterium source;

    Get apart from the oil-polluted soils at oil production waste water in oil field discharge outlet place as oil degradation original inhabitants bacterium bacterium source;

    Get apart from the oil extraction waste water of oil production waste water in oil field treatment plant water outlet and sludge sewage as halophilic bacterium bacterium source;

    Step 2;

    the screening of oil degradation original inhabitants bacterium;

    (1) get soil-like 2g as oil degradation original inhabitants bacterium bacterium source, add and fill the Erlenmeyer flask of 100mL sterilized water, and place shaking culture case room temperature vibration 0.5~

    2h, oscillation frequency is 200rpm;

    (2) get 5mL solution from the Erlenmeyer flask of sterilized water, add shake-flask culture among the 100mL gradient screening and culturing liquid A, described gradient screening and culturing liquid A is the mixing solutions of mass concentration 75% beef-protein medium and 1g/L crude oil;

    (3) when gradient screening and culturing liquid A is muddy, get 5mL and be forwarded to shake-flask culture among the 100mL gradient screening and culturing liquid B from gradient screening and culturing liquid A, described gradient screening and culturing liquid B is that mass concentration is the mixing solutions of 50% beef-protein medium and 2g/L crude oil;

    (4) when gradient screening and culturing liquid B is muddy, get 5mL and be forwarded to shake-flask culture among the 100mL gradient screening and culturing liquid C from gradient screening and culturing liquid B, described gradient screening and culturing liquid C is that mass concentration is the mixing solutions of 25% beef-protein medium and 3g/L crude oil;

    (5) when gradient screening and culturing liquid C is muddy, gets 5mL and be forwarded to shake-flask culture in the 100mL minimal medium from gradient screening and culturing liquid C, when minimal medium became muddy, obtaining with the oil was the oil degradation original inhabitants bacterium of sole carbon source, and refrigeration;

    Step 3;

    the screening of halophilic bacterium;

    Get each 1ml of active sludge and oil extraction waste water, adopt the 100mL beef-protein medium to cultivate, obtaining salinity through the gradient acclimation and screening is 1%~

    20% halophilic bacterium composite microbial system, and utilizes the halophilic bacterium composite microbial system to prepare cell concentration 10 8

    10 9The mixing halophilic bacterium nutrient solution of individual/ml;

    Step 4;

    the screening of oil degradation halophilic bacterium;

    (1) have a liking for the screening that salinity is the nutrient solution of 1.0% oil degradation halophilic bacterium;

    (A) get soil-like 2g, add and fill in the Erlenmeyer flask of 100mL sterilized water, and place the vibration of shaking culture case room temperature as oil degradation original inhabitants bacterium bacterium source;

    (B) from the Erlenmeyer flask of sterilized water, get 5mL, add among the 100mL enrichment culture liquid A, and in wherein add mixing halophilic bacterium nutrient solution 5mL shake-flask culture, described enrichment culture liquid A is the mixing solutions of mass concentration 75% beef-protein medium, 1g/L crude oil and 1.0%NaCl;

    (C) when enrichment culture liquid A is muddy, therefrom gets 5mL and be forwarded to shake-flask culture among the 100mL enrichment culture liquid B, described enrichment culture liquid B is the mixing solutions of mass concentration 50% beef-protein medium, 2g/L crude oil and 1.0%NaCl;

    (D) when enrichment culture liquid B is muddy, therefrom gets 5mL and be forwarded to shake-flask culture among the 100mL enrichment culture liquid C, described enrichment culture liquid C is the mixing solutions of mass concentration 25% beef-protein medium, 3g/L crude oil and 1.0%NaCl;

    (E) when enrichment culture liquid C is muddy, therefrom get 5mL and be forwarded to shake-flask culture among the 100mL inorganic salt enrichment culture liquid A, described inorganic salt enrichment culture liquid A is that minimal medium and mass concentration are the mixing solutions of 1.0%NaCl, when inorganic salt enrichment culture liquid A became muddy, obtaining with the oil was the nutrient solution that salinity is 1.0% oil degradation halophilic bacterium of having a liking for of sole carbon source;

    (2) from have a liking for the nutrient solution that salinity is 1.0% oil degradation halophilic bacterium, get 5ml, be forwarded to shake-flask culture among the 100mL inorganic salt enrichment culture liquid B, when inorganic salt enrichment culture liquid B becomes muddy, obtain having a liking for the nutrient solution that salinity is the oil degradation halophilic bacterium of X;

    Described inorganic salt enrichment culture liquid B is that minimal medium contains the NaCl solution that mass concentration is X, and 1.0%<

    X<

    10.0%;

    (3) from have a liking for the nutrient solution of oil degradation halophilic bacterium that salinity is X, get 5ml, be forwarded to shake-flask culture among the 100mL inorganic salt enrichment culture liquid C, when inorganic salt enrichment culture liquid C becomes muddy, obtain having a liking for the nutrient solution that salinity is the oil degradation halophilic bacterium of Y;

    Described inorganic salt enrichment culture liquid C is that minimal medium contains the NaCl solution that mass concentration is Y, and 1.0%<

    X<

    Y<

    10.0%;

    (4) from have a liking for the nutrient solution of oil degradation halophilic bacterium that salinity is Y, get 5ml, be forwarded to shake-flask culture among the 100mL inorganic salt enrichment culture liquid D, when inorganic salt enrichment culture liquid D becomes muddy, obtain having a liking for the nutrient solution that salinity is the oil degradation halophilic bacterium of Z;

    Described inorganic salt enrichment culture liquid D is that minimal medium contains the NaCl solution that mass concentration is Z, and 1.0%<

    X<

    Y<

    Z<

    10.0%;

    (5) from have a liking for the nutrient solution of oil degradation halophilic bacterium that salinity is Z, get 5ml, be forwarded to shake-flask culture among the 100mL inorganic salt enrichment culture liquid E, when inorganic salt enrichment culture liquid E becomes muddy, obtain having a liking for the nutrient solution that salinity is the oil degradation halophilic bacterium of E;

    Described inorganic salt enrichment culture liquid E is that to contain mass concentration be 10.0% NaCl solution to minimal medium;

    (6) salinity is 1.0% for having a liking for of will obtaining, X, Y, Z and 10.0% oil degradation halophilic bacterium mix according to the equal volume ratio, obtains oil degradation halophilic bacterium composite fungus agent;

    Step 5;

    the high-effect bacterial screening that oil-polluted soils is repaired;

    (1) the dull and stereotyped activation of the corresponding salinity of the oil degradation halophilic bacterium composite fungus agent utilization that obtains in the step 3, and with step 2 in the oil degradation original inhabitants bacterium that obtains adopt respectively the beef-protein medium shaking culture to cell concentration all greater than every milliliter 10 9Individual, centrifugal collection thalline prepares two kinds of zymocyte liquids;

    (2) get soil as oil degradation original inhabitants bacterium bacterium source, through removal of impurities, air-dry, fragmentary, cross 200 mesh sieves, mixing after, soil is tiled into thin layer;

    (3) soil is respectively charged in the flowerpot, measures the oleaginousness and the saltiness of soil in the flowerpot, regulate pH in soil and humidity;

    In flowerpot, inoculate the zymocyte liquid of oil degradation original inhabitants bacterium and the zymocyte liquid of oil degradation halophilic bacterium composite fungus agent respectively;

    Throwing bacterium amount is in every gram soil and contains 10 6

    10 8Individual bacterium colony, and added once in per 10~

    15 days;

    (4) in flowerpot, added the one time of nutrition material every 10~

    15 days, abundant mixing, and regulate pH in soil and humidity every day;

    (5) survey oleaginousness in each flowerpot by weighting method, utilize the plate technique method to measure the concentration of thalline, measure and also regulate pH in soil, behind 7 time-of-weeks, measure the degradation rate of soil PetroChina Company Limited. in each flowerpot, obtain the highest high-effect bacterial of degradation rate;

    Step 6;

    the reparation of high-effect bacterial;

    Utilize the quadrature reparation to test and choose the bacterium amount of throwing, humidity respectively, add sucrose amount and saltpetre amount as the repairing condition of high-effect bacterial, wherein throw the bacterium amount and add 10 in every gram soil 6

    10 8Individual thalline, humidity is 20%~

    40%, and adding the sucrose amount is every kilogram of soil 1~

    10g, and adding the saltpetre amount is every kilogram of dry ground 2~

    16g;

    Obtain oil-polluted soils biological restoration microbial inoculum at last.

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